In medical and bioscience experiments, cells as an observation object have been imaged by a CCD camera or the like and converted into data, and various image processing techniques have been applied to this image data for observation and analysis. Conventionally, cells have been dyed using a dye to enhance the visibility of the cells. To suppress damage given to the cells, it is required to image the cells without dyeing them in some cases. However, since cytoplasm of the undyed cells are almost colorless and transparent, it is difficult to obtain an image with high quality. Particularly, it is difficult to obtain an image with a high contrast in bright field imaging.
A technique for imaging undyed cells using a phase difference microscope is disclosed as a technique for imaging substantially transparent cells in patent literature 1. In this technique, a difference image of two contrast images having mutually different signs of a phase difference is generated from those images. Specifically, a difference of images imaged in a defocused manner by a very short distance from an in-focus position toward a near point side and a far point side is obtained. Thus, it is a premise to perform imaging after the in-focus position is grasped. However, a method for adjusting a focus position for a substantially transparent object is not described in patent literature 1.
It is thought to be possible to apply, for example, a technique described in patent literature 2 as a method for obtaining an in-focus position in this case. In this technique, imaging is performed while a focus position is changed at equal intervals. A position where a contrast value is maximized in a difference image of two images having adjacent focus positions is set as an in-focus position.